4-substituted-3-(2-amino-2-cycloalkyl methyl)-acetamido azetidin-2-one derivatives as cysteine proteinase regulators

ABSTRACT

In accordance with the present invention, there are provided 4-substituted-3-(2-amino-2-cycloalkyl methyl)-acetamido azetidin-2-one derivatives of the formula: ##STR1## wherein n is 1, 2 or 3; in which R 1 , R 2  and R 3  are as defined herein, and salts thereof, which exhibit excellent cysteine proteinase inhibitory activity and which can be used for treatment of different diseases such as muscular dystrophy, myocardial infarction, bone resorption, arthritis, cancer metastasis, pulmonary emphysema, septic shock, cerebral ischemia, memory function, Alzheimer and cataract, malaria, glomerular basement membrane degradation, bacterial infection, inflammatory diseases, parasitic infections, and viral infections.

This application claims priority of U.S. Provisional patent applicationSer. No. 60/026,514, filed Sep. 23, 1996.

BACKGROUND OF THE INVENTION

Cysteine proteinases containing a highly reactive cysteine residue witha free thiol group at the active site have been known as playing animportant role in certain conditions distinguished by aberrant proteinturnover such as: muscular dystrophy (Am. J. Pathol. 1986, 122, 193-198;Am. J. Pathol. 1987, 127, 461-466), myocardial infarction (J. Am. Coll.Cardiol. 1983, 2, 681-688), bone resorption (Biochem. J. 1991, 279,167-274; J. Biol. Chem. 1996, 271, 2126-2132; and Biochem. Biophys. Acta1992, 1116, 57-66), arthritis (Arthritis Rheumatism 1994, 37, 236-247;and Biochem. Pharmacol. 1992, 44, 1201-1207), cancer metastasis (CancerMetastasis Rev. 1990, 9, 333-352), pulmonary emphysema (Am. Rev. Respir.Dis. 1975, 111, 579-586), septic shock (Immunol. Today 1991, 11,404-410, Biochemistry 1994, 33, 3934-3940), cerebral ischemia, memoryfunction, Alzheimer and cataract (TIPS 1994, 15, 412-419, Bioorg. Med.Chem. Lett. 1995, 4, 387-392, Proc. Natl. Acad. Sci. USA 1991, 88,10998-11002), malaria (J. Med. Chem. 1995, 38, 5031-5037), glomerularbasement membrane degradation (Biochem Bioph. Acta 1989, 990, 246-251),bacterial infection (Nature 1989, 337, 385-386), inflammatory diseases(Protein Science 1995, 4, 3-12), parasitic infections (Annu. Rev.Microbiol. 1993, 47, 821-853; Parasitol. Today 1990, 6, 270-275), andviral infections (Biochem. 1992, 31, 7862-7869).

A variety of cysteine proteinases have been shown to be present inmammalian tissue. The most notable of these proteinases are thelysosomal cathepsins (cathepsin B, H, S, L and K) and the cytoplasmicCa⁺² dependent enzymes, the calpains. These enzymes are, therefore,excellent targets for specific inhibitors as therapeutic agents for theconditions such as those noted above.

Cysteine proteinases are inhibited by several types of peptide derivedinhibitors such as peptidyl aldehyde (Eur. J. Biochem. 1982, 129,33-41), chloromethyl ketone (Acta. Biol. Med. Ger. 1981, 40, 1503-1511),diazomethyl ketone (Biochemistry 1977, 16, 5857-5861), monofluoromethylketone (Biochemical Pharmacology 1992 44, 1201-1207), acyloxy methylketone (J. Med. Chem. 1994, 37, 1833-1840), O-acyl hydroxamates(Biochem. Biophy. Research Communications 1988, 155, 1201-1206), methylsulphonium salts (J. Biol. Chem. 1988, 263, 2768-2772) and epoxysuccinyl derivatives (Agric. Biol. Chem. 1978, 42, 523-527) withoutsignificantly inhibiting other classes of proteinases.

SUMMARY OF THE INVENTION

Our laboratory has been extensively involved in search for novelcysteine proteinase regulators and found that4-substituted-3-(amino-2-cycloalkyl methyl)-acetamido azetidin-2-onederivatives exhibit excellent activity and selectivity within the classof cysteine proteinases. There is an ongoing need to improve in vivoefficacy by improving plasma stability and pharmacokinetics. ##STR2##

The molecular modelling of 3-substituted phenyl alanyl azetidinonesuggested that the replacement of phenyl alanine with cyclohexyl alaninemight increase the hydrophobic binding with cysteine proteinases.Unfortunately, there is no increase in activity as expected but itshowed improved stability in plasma and good in vivo activity. Thisfinding of novel 4-substituted-3-(2-amino-2-cycloalkyl methyl)-acetamidoazetidin-2-one derivatives is reported in the present invention ascysteine proteinase inhibitors.

In accordance with the present invention, there are provided4-substituted-3-(2-amino-2-cycloalkyl methyl)-acetamido azetidin-2-onederivatives which exhibit cysteine proteinase regulatory (e.g.,inhibitory) activity with improved stability in biological fluids andwhich can be used for treatment of different diseases such as musculardystrophy, myocardial infarction, bone resorption, arthritis, cancermetastasis, pulmonary emphysema, septic shock, cerebral ischemia, memoryfunction, Alzheimer and cataract, malaria, glomerular basement membranedegradation, bacterial infection, inflammatory diseases, parasiticinfections, and viral infections.

In accordance with the present invention, there are provided compoundsof formula I and pharmaceutically acceptable salts thereof: ##STR3##wherein n is 1, 2 or 3;

R₁ is

hydrogen; or

--SO₃ ⁻ M⁺ wherein M is a hydrogen atom, a metal ion which is selectedfrom sodium, potassium, magnesium, and calcium, or N⁺ (R₄)₄ wherein R₄is a C₁ -C₆ alkyl group;

R₂ is

(a) a group --OCOR₅ wherein R₅ is

(i) a C₁ -C₆ alkyl group,

(ii) a C₂ -C₆ alkenyl group,

(iii) a C₂ -C₆ alkynyl group,

(iv) a C₃ -C₆ cycloalkyl group,

(v) a phenyl group,

(vi) a naphthyl group, or

(vii) a monocyclic or bicyclic heterocyclic group, which group (i),(ii), (iii), (iv), (v), (vi), or (vii) is unsubstituted or substitutedby 1, 2 or 3 substituents independently selected from

hydroxy,

halogen,

carboxy,

C₁ -C₄ alkyl (which is unsubstituted or substituted at least once withcarboxy and/or amino),

C₁ -C₂ alkoxy,

amino,

cyano, and

phenyl and monocyclic or bicyclic heterocyclic groups, which phenyl andheterocyclic groups are unsubstituted or substituted by 1 or 2substituents independently selected from

hydroxy,

halogen,

carboxy,

C₁ -C₄ alkyl,

C₁ -C₂ alkoxy,

amino, and

cyano;

or (b) a group --XR₅ wherein X is selected from the group consisting ofO, S, SO, and SO₂, and R₅ is as defined above;

R₃ is hydrogen, --COOR₅, --COR₅, --SO₂ R₅, or --COR₁₄ wherein R₅ is asdefined above and R₁₄ is amino group which is unsubstituted orsubstituted at least once with C₁ -C₆ alkyl group which is unsubstitutedor substituted at least once with 1 or 2 substitutents selected fromhydroxy, halogen, cyano, amino, heterocycle, and phenyl (wherein theheterocycle or phenyl is unsubstituted or substituted at least once by 1or 2 substituents selected from halogen, hydroxy, cyano, carboxy andamino).

In accordance with a preferred aspect of the present invention, thereare provided compounds of formula I and pharmaceutically acceptablesalts thereof:

wherein:

n is 1, 2 or 3

R₁ is

hydrogen;

--SO₃ ⁻ M⁺ wherein M is a hydrogen atom, a metal ion which is selectedfrom sodium, potassium, magnesium, or calcium, or N⁺ (R₄)₄ wherein R₄ isa C₁ -C₆ alkyl group;

R₂ is

--OCOR₅ wherein R₅ is

(i) a C₁ -C₆ alkyl group which is unsubstituted or substituted at leastonce by 1 or 2 substitutents selected from hydroxy, halogen, and amino;or

(ii) a phenyl group which is unsubstituted or substituted at least onceby 1-3 substituents selected from hydroxy, halogen, C₁ -C₄ alkyl group,C₁ -C₂ alkoxy group, and cyano;

--XR₆ wherein X is O, S, SO, or SO₂ ; R₆ is

(i) a C₁ -C₆ alkyl group which is unsubstituted or substituted at leastonce by 1 or 2 substitutents selected from hydroxy, halogen, amino orphenyl; or

(ii) a phenyl group which is unsubstituted or substituted at least onceby 1-3 substituents selected from hydroxy, halogen, carboxy, and C₁ -C₄alkyl group which is unsubstituted or substituted at least once withcarboxy, amino or both, C₁ -C₂ alkoxy group, cyano or heterocycle group;

R₃ is

hydrogen;

--COOR₇ wherein R₇ is a C₁ -C₆ alkyl group which is unsubstituted orsubstituted at least once with phenyl and/or heterocycle group;

--COR₈ wherein R₈ is

(i) a C₁ -C₆ alkyl group which is unsubstituted or substituted at leastonce by 1 or 2 substitutents selected from hydroxy, halogen, cyano,amino, heterocycle, and phenyl, wherein said heterocycle or phenyl isunsubstituted or substituted at least once by 1 or 2 substituentsselected from halogen, hydroxy, cyano, carboxy and amino; or

(ii) an amino group which is unsubstituted or substituted at least oncewith C₁ -C₆ alkyl group which is unsubstituted or substituted at leastonce by 1 or 2 substitutents selected from hydroxy, halogen, cyano,amino, heterocycle and phenyl, wherein said heterocycle or phenyl isunsubstituted or substituted at least once by 1 or 2 substituentsselected from halogen, hydroxy, cyano, carboxy and amino; or

--SO₂ R₉ wherein R₉ is

(i) a C₁ -C₆ alkyl group which is unsubstituted or substituted at leastonce with heterocycle and/or phenyl; or

(ii) a C₂ -C₄ alkenyl group which is unsubstituted or substituted atleast once with heterocycle and/or phenyl.

The pharmaceutically acceptable salts of formula I are selected fromsalts of sodium, potassium, magnesium, calcium, hydrogen chloride,tartaric acid, succinic acid, fumaric acid or p-toluenesulfonic acid.

Examples of C₁ -C₆ alkyl group as substituents in R₄, R₅, R₆, R₈, or R₉are straight or branched chain alkyl group having 1-6 carbon atoms suchas methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl,2-methylprop-1-yl, 2-methylprop-2-yl, pentyl, 3-methylbutyl, hexyl andthe like.

Examples of halogen atoms as substitutents in R₅, R₆, or R₉ arefluorine, chlorine, bromine or iodine.

Examples of C₂ -C₄ alkenyl group as defined in R₉ are alkenyl grouphaving 2-4 carbon atoms such as ethenyl, 1-propenyl, 2-propenyl,1-butenyl, 3-butenyl and the like.

Suitable heterocyclic groups in accordance with the present inventioninclude 5- or 6-membered aromatic or non-aromatic heterocyclic groupscontaining 1, 2, 3 or 4 heteroatoms selected from O, S or N, andbicyclic heterocyclic groups including a monocyclic heterocyclic asdefined above which is fused to a second 5- or 6-membered carbocyclic or5- or 6-membered heterocyclic ring.

Examples of heterocyclic group as defined in R₅, R₆, R₇, R₈ or R₉ are C₂-C₉ mono or bicyclic heterocyclic group which may have 1-3 heteroatomsselected from nitrogen, sulphur or oxygen such as thiophene, pyridine,1,2,3-triazole, 1,2,4-triazole, quinoline, benzofuran, benzothiophene,morpholine, thiomorpholine, piperizine, piperidine and the like.

Examples of C₁ -C₄ alkyl group as substituents in R₅, R₆, or R₉ aremethyl, ethyl, propyl, 2-methyl propyl, butyl, 1,1-dimethyl ethyl andthe like.

Examples of C₁ -C₂ alkoxy group as substituents in R₅, R₆, or R₉ aremethoxy or ethoxy.

The azetidinone nucleus carries two asymmetric carbon atoms at position3 and 4, and can exist as 4-diastereoisomers. In general, the preferredisomer is that in which the hydrogen atoms at C3 and C4 are cis to eachother for superior inhibitory activity against different cysteineproteinase such as papain, Cathepsin B, Cathepsin H, Cathepsin K andCathepsin L. Such diastereoisomers and their racemic mixtures are alsoincluded within use of the azetidinone derivatives as cystein proteinaseinhibitor.

In accordance with preferred embodiments of the invention, there areprovided 4-substituted-3-(2-amino-2-cycloalkyl methyl)-acetamidoazetidin-2-one derivatives of formula I: ##STR4## wherein: n is 1, 2 or3

R₁ is selected from hydrogen, or sulphonic acid;

R₂ is selected from acetoxy, butyloxy, 2-carboxy ethyloxy,2-aminoethyloxy, 2-fluoro ethoxy, phenoxy, methyl phenoxy, morpholinophenyloxy, 2-hydroxy ethylthio, phenylthio, phenylsulphonyl,4-(2-carboxy-2-amino ethyl)-phenoxy, 4-carboxy phenoxy, 3-carboxyphenoxy, 2-pyridylthio, 4-pyridylthio, benzyloxy and the like; and

R₃ is selected from alkanoyl, aryloxy carbonyl, 3-aryl propanoyl,3-heteroaryl propanoyl, arylmethylaminocarbonyl,2-aryl-eth-1-en-sulphonyl, and the like.

Preferred embodiments of the present invention include the followingcompounds:

(3S,4S)-3-(2S-2-benzyloxycarbonylamino-2-cyclohexylmethyl-acetamido)-4-acetoxy-azetidin-2-one;

(3S,4S)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one;

(3S,4S)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-{4-(2S-2-amino-2-carboxy-ethyl)-phenoxy}-azetidin-2-one;

(3S,4R)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-{4-(2S-2-amino-2-carboxy-ethyl)-phenoxy}-azetidin-2-one;

(3S,4SR)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-phenylthio-azetidin-2-one;

(3S,4SR)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-phenylsulfonyl-azetidin-2-one;

(3S,4S)-3-{2S-2-(benzylaminocarbonyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one;

(3S,4S)-3-{2S-2-(phenylethenylsulfonyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one;

(3S,4S)-3-(2S-2-benzyloxycarbonylamino-2-cyclohexylmethyl-acetamido)-4-(3-methyl-phenoxy)-azetidin-2-one;

(3S,4R)-3-(2S-2-benzyloxycarbonylamino-2-cyclohexylmethyl-acetamido)-4-(3-methyl-phenoxy)-azetidin-2-one;

(3S,4S)-3-{2S-2-3-(pyridin-4-yl)propenoyl!amino-2-cyclohexylmethyl-acetamido}-4-phenoxy-azetidin-2-one;and

(3S,4S)-3-{2S-2-3-(pyridin-3-yl)propenoyl!amino-2-cyclohexylmethyl-acetamido}-4-phenoxy-azetidin-2-one.

Compounds of formula I may be utilized for different diseases such asmuscular dystrophy, myocardial infarction, bone resorption, arthritis,cancer metastasis, pulmonary emphysema, septic shock, cerebral ischemia,memory function, Alzheimer and cataract, malaria, glomerular basementmembrane degradation, bacterial infection, inflammatory diseases,parasitic infections, and viral infections by regulating the cysteineproteinases in medicaments formulated with pharmaceutically acceptablecarriers.

BRIEF DESCRIPTION OF THE DRAWING FIGURE

The FIGURE is a graph of in vitro stability of compound 3 (see Example3) and a reference compound in rat plasma.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to certain3,4-disubstituted-azetidin-2-one derivatives having cysteine proteinaseinhibitory activity and stability in biological fluids. The compounds ofthis invention include compounds having hydrogen, ester (OCOR₅), ether(OR₅), thioether (SR₅), sulfone (SO2R₅) and sulfoxide (SOR₅) at position4 and cycloalkyl alanine group at position 3 of 3-amino-azetidin-2-one(II). Certain derivatives of formula I are prepared by the commonintermediates II by reacting with cycloalkyl alanine either in presenceof dicyclohexylcarbidiimide (DCC) or acid chloride in presence of base,or activated ester according to techniques known in the art. ##STR5##

The preparation of compounds II is carried out by following thesynthetic route as described in Eur. J. Med. Chem 1992, 27, 131-140, andTetrahedron 1983, 39, 2577-2589, wherein R₂ is OCOR₅, and R₃ is asubstituent group COOR₇. The definitions of R₁, R₅ and R₇ are the sameas defined above.

Certain 4-substituted-3-(2-amino-2-cycloalkyl methyl)-acetamidoazetidin-2-one derivatives of formula I wherein substititions at theamino acid group are other than COOR₅, such as COR₅ or SO₂ R₅ areprepared by following the synthetic route as shown in the schemedepicted below. The R₅ groups are the same as defined above. ##STR6##

The benzyloxycarbonyl cyclohexyl alanine are desubstituted andresubstituted through amide bond by reacting with R₅ --COOH either inpresence of DCC or acid chloride in presence of base or anhydride inpresence of base or activated ester, or through sulphonamide bond byreacting with R₅ SO₂ Cl in presence of base or through urea bond byreacting with R₅ NCO. R₁₁ is a C₁ -C₆ alkyl group which is unsubstitutedor substituted with phenyl or heterocyclic group.

Certain 4-substituted-3-(2-amino-2-cycloalkyl methyl)-acetamidoazetidin-2-one derivatives of formula I wherein R₂ is XR₅, wherein X isO or S, and R₅ is the same as defined above, are prepared by followingthe synthetic route as shown below starting from compound of formula Iwherein R₂ is OCOCH₃ by reacting with R₅ XH in presence of Lewis acidssuch as zinc acetate, zinc iodide, zinc chloride, titaniumtetrachloride, palladium acetate, boron trifluoride, aluminumtrichloride and the like or in presence of base such as sodiumhydroxide. There are cases where carboxy group as substituent in R₅ issubstituted with R₁₁ such as diphenyl methyl or 1,1-dimethyl ethyl, oramino group as substituent in R₅ is substituted with R₁₂ such asbenzyloxy carbonyl or 1,1-dimethyl ethoxy carbonyl, or both groups assubstituents in R₅ together are desubstituted by hydrogenation orhydrolysis with acids. ##STR7##

Certain 4-substituted-3-(2-amino-2-cyclo-alkyl methyl)-acetamidoazetidin-2-one derivatives of formula I wherein R₂ is SR₅ are convertedto SOR₅ or SO₂ R₅ by oxidation with oxidizing agent selected fromm-chloroperbenzoic acid, hydrogen peroxide, peracetic acid, potassiumpermanganate, magnesium dioxide and the like. The synthetic route isoutlined below. ##STR8##

4-substituted-3-(2-amino-2-cycloalkyl methyl)-acetamido azetidin-2-onederivatives of formula I wherein R₁ is hydrogen can be converted toN-sulphonic acid by the sulphonation with pyridine-SO₃ ordimethylformamide-SO₃ complex by following the synthetic route asoutlined below. ##STR9##

In the above descriptions, the reactants are reacted together withsolvent at elevated or low temperatures for sufficient time to allow thereaction to proceed to completion. The reaction conditions will dependupon the nature and reactivity of the reactants. Wherever a base is usedin a reaction, they are selected from triethylamine, pyridine,4-dimethylaminopyridine, diisopropylethylamine, 1,5-diazabicyclo4,3,0!non-5-ene, 1,8-diazabicyclo 5,4,0!undec-7-ene, sodium carbonate,potassium carbonate or cesium carbonate.

The solvent of choice for the reaction is selected from non-reactivesolvents depending on the reactants such as benzene, toluene,acetonitrile, tetrahydrofuran, ethanol, methanol, chloroform, ethylacetate, methylene chloride, dimethyl formamide, dimethyl sulphoxide,hexamethyl phosphoric triamide, or the like. Solvent mixtures may alsobe utilized.

Suitable reaction temperatures are generally in the range of from -70°C. to 150° C. The preferred molar ratio of reactants is 1:1 to 1:5. Thereaction time is in the range of from 0.5 to 72 hours, depending on thereactants.

The desubstitution of N-substituent group is carried out either byhydrogenation or by hydrolysis with appropriate acids such ashydrochloric acid, trifluoroacetic acid or acetic acid in solvent suchas methanol, ethanol, propanol or ethyl acetate. The hydrogenationreaction is usually carried out in the presence of a metal catalyst,such as Pd, Pt, or Rh, under normal pressure to high pressure.

The compounds of this invention, when used alone or in combination withother drugs as an agent for treating muscular dystrophy, osteoporosis orcancer metastasis in mammals including humans, may take pharmaceuticaldosage forms including parenteral preparations such as injections,suppositories, aerosols and the like, and oral preparations such astablets, coated tablets, powders, granules, capsules, liquids and thelike. Injections are generally preferred. The above preparations areformulated in a manner known in the art.

For the formulation of solid preparations for oral administration, anexcipient, and if desired, a binder, disintegrator, lubricant, coloringagent, corrigent, flavor etc. are added to the compound of theinvention, and then tablets, coated tablets, granules, powders, capsulesor the like are prepared in a conventional manner.

For the formulation of injections, a pH adjusting agent, buffer,stabilizer, isotonic agent, local anesthetic or the like is added to theactive ingredient of the invention, and injections for subcutaneous,intramuscular or intravenous administration can be prepared in theconventional manner.

For the formulation of suppositories, a base, and if desired, asurfactant are added to the active ingredient of the invention, and thesuppositories are prepared in a conventional manner.

The excipients useful for solid preparations for oral administration arethose generally used in the art, and the useful examples are excipientssuch as lactose, sucrose, sodium chloride, starches, calcium carbonate,kaolin, crystalline cellulose, methyl cellulose, glycerin, sodiumalginate, gum arabic and the like, binders such as polyvinyl alcohol,polyvinyl ether, polyvinyl pyrrolidone, ethyl cellulose, gum arabic,schellac, sucrose, water, ethanol, propanol, carboxymethyl cellulose,potassium phosphate and the like, lubricants such as magnesium stearate,talc and the like, and further include additives such as usual knowncolouring agents, disintegrators and the like. Examples of bases usefulfor the formulation of suppositories are oleaginous bases such as cacaobutter, polyethylene glycol, lanolin, fatty acid triglycerides, witepsol(trademark, Dynamite Nobel Co. Ltd.) and the like. Liquid preparationsmay be in the form of aqueous or oleaginous suspension, solution, syrup,elixir and the like, which can be prepared by a conventional way usingadditives.

The amount of the compound I of the invention to be incorporated intothe pharmaceutical composition of the invention varies with the dosageform, solubility and chemical properties of the compound, administrationroute, administration scheme and the like. Preferably, the amount isabout 1 to 25% (w/w) in the case of oral preparations, and about 0.1 toabout 5% (w/w) in the case of injections which are parenteralpreparations.

The dosage of the compound I of the invention is suitably determineddepending on the individual cases taking symptoms, age and sex of thesubject and the like into consideration. Usually the dosage in the caseof oral administration is about 50 to 1500 mg per day for an adult in 2to 4 divided doses, and the dosage in the case of injection, forexample, by intravenous administration is 2 ml (about 1 to 100 mg) whichis administered once a day for adults wherein the injection may bediluted with physiological saline or glucose injection liquid if sodesired, and slowly administered over at least 5 minutes. The dosage incase of suppositories is about 1 to 1000 mg which is administered onceor twice a day at an interval of 6 to 12 hours wherein the suppositoriesare administered by insertion into the rectum.

EXAMPLE 1(3S,4S)-3-(2S-2-benzyloxycarbonylamino-2-cyclohexylmethyl-acetamido)-4-acetoxy-azetidin-2-one(1)

(3S,4S)-3-benzyloxycarbonylamino-4-acetoxy-azetidin-2-one (912 mg, 3.28mmol) is hydrogenated with 1 g of 10% palladium on activated carbon in35 ml of ethyl acetate at 50 psi hydrogen pressure at room temperaturefor 1.5 hours. After removal of catalyst by filtration, desubstitution(3S,4S)-3-amino-4-acetoxy-azetidin-2-one in ethyl acetate is obtained.

To a solution of 2S-2-benzyloxycarbonylamino-2-cyclohexylmethyl-aceticacid (1.0 g, 3.28 mmol) and 1-hydroxy-benzotriazole (443 mg, 3.28 mmol)in THF (30 ml), DCC (676 mg, 3.28 mmol)/THF (10 ml) is added at 0° C.The reaction mixture is stirred at room temperature for 2 hours and thencooled with an ice bath. The resulting DCU is removed by filtration.Then, a precooled solution of (3S,4S)-3-amino-4-acetoxy-azetidin-2-onein ethyl acetate is added at -15° C. and the resulting mixture isstirred at a bath temperature of -15 to 5° C. for 1 hour and then atroom temperature for 3 hours. After removal of solvent, the residue isdissolved in ethyl acetate, washed with cold saturated NaHCO₃ solution,water, brine and dried over sodium sulphate. After removal of solvent,the residue is purified by silica gel column chromatography usinghexane-ethyl acetate (1:1) as eluent and the title compound is obtained.

Yield: 92%, m.p.: 134-135° C., FAB-MS: 432 (MH⁺), calcd for C₂₂ H₂₉ N₃O₆ 431

¹ H NMR (DMSO-d₆), δ (ppm): 0.75-1.8 (13 H, m), 2.08 (3H, s), 4.00-4.15(1H, m), 4.64 (1H, d, J=8 Hz), 5.04 (2H, m), 5.75 (1H, s), 7.30-7.45(5H, m), 7.48 (1H, d, J=8 Hz), 8.67 (1H, d, J=8.3 Hz), 9.16 (1H, s).

IR (KBr, cm⁻¹): 3325, 2925, 1797, 1747, 1693, 1661, 1536, 1446, 1371,1270, 1227.

EXAMPLE 2(3S,4S)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one(2)

By a similar method as described in example 1, the title compound isobtained by reacting2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetic acid with(3S,4S)-3-amino-4-acetoxy-azetidin-2-one.

Yield: 85%, m.p.: 166-168° C. (dec.), FAB-MS: 430 (MH⁺), calcd for C₂₃H₃₁ N₃ O₅ 429

¹ H NMR (CDCl₃ -d₆), δ (ppm): 0.80-1.80 (13H, m), 2.10 (3H, s), 2.53(2H, t, J=7.5 Hz), 2.94 (2H, t, J=7.5 Hz), 4.54 (1H, m), 4.62 (1H, d,J=7.5 Hz), 5.80 (1H, s), 6.18 (1H, d, J=8.1 Hz), 7.10-7.35 (6H, m), 7.54(1H, d, J=7.5 Hz).

IR (KBr, cm⁻¹): 3275, 2925, 1794, 1739, 1656, 1634, 1531, 1440, 1358,1219.

EXAMPLE 3(3S,4S)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-{4-(2S-2-amino-2-carboxy-ethyl)-phenoxy}-azetidin-2-one(3)

A mixture of(3S,4S)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one(550 mg, 1.28 mmol),4-(2S-2-N-benzyloxycarbonylamino-2-diphenylmethoxycarbonyl-ethyl)-phenol(481 mg, 1 mmol), and zinc acetate dihydrate (300 mg, 1.36 mmol) in amixture of benzene (18 ml) and toluene (18 ml) is refluxed for 5 hoursusing Dean-Stark water separator. The reaction mixture is purified bysilica gel column chromatography using hexane-ethyl acetate as eluentand 200 mg of(3S,4S)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-{(2S-2-N-benzyloxy-carbonylamino-2-diphenylmethoxycarbonyl-ethyl)-phenoxy}-azetidin-2-oneis obtained.

¹ H NMR (CDCl₃ -d₆), δ (ppm): 0.80-1.80 (13H, m), 2.45 (2H, t, J=7.5Hz), 2.87 (2H, t, J=7.5 Hz), 3.01 (2H, m), 4.45-4.70 (3H, m), 5.03 (2H,s), 5.60 (1H, s), 6.50-6.90 (6H, m), 7.1-7.4 (21H, m), 7.58 (1H, d,J=7.5 Hz).

200 mg of(3S,4S)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-{(2S-2-N-benzyloxy-carbonylamino-2-diphenylmethoxycarbonyl-ethyl)-phenoxy}-azetidin-2-one ishydrogenated with 500 mg of 10% palladium on activated carbon in 50 mlof ethyl acetate at 50 psi hydrogen pressure at room temperature for 2.5hours. The solid is filtered and washed with ethyl acetate (3×10 ml).The solid is extracted with a mixture of water/acetonitrile (3:7) (3×20ml). After removal of solvent, 31 mg of the title compound is obtainedas white solid.

Yield: 24%, m.p.: 180° C. (dec.), FAB-MS: 551 (MH⁺), calcd for C₃₀ H₃₈N₄ O₆ 550

¹ H NMR (DMSO-d₆), δ (ppm): 0.7-1.8 (13H, m), 2.35-2.55 (2H, m),2.70-2.90 (2H, m), 3.20-3.40 (2H, m), 4.29 (1H, m), 4.65 (1H, d, J=8Hz), 5.49 (1H, s), 6.83 (2H, m), 7.15-7.35 (7H, m), 8.10 (1H, d, J=8Hz), 8.75 (1H, d, J=8 Hz), 9.32 (1H, s).

IR (KBr, cm⁻¹): 3385, 2925, 1791, 1750, 1681, 1647, 1623, 1556, 1522,1384, 1227.

EXAMPLE 4(3S,4R)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-{4-(2S-2-amino-2-carboxy-ethyl)-phenoxy}-azetidin-2-one(4)

To a solution of 4-(2S-2-N-benzyloxycarbonylamino-2-diphenylmethoxycarbonyl-ethyl)-phenol (7.46 g, 15.6 mmol) in acetone (80 ml), H₂ O (20ml) and 1 N NaOH (14 ml),(3S,4S)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one(5.51 g, 12.8 mmol) in acetone (100 ml) and H₂ O (50 ml) is slowly addedat 5° C. The mixture is stirred at 5° C. for 2 hours. After removal ofsolvent, the residue is dissolved in ethyl acetate, washed with water,brine and dried over sodium sulphate. After removal of solvent, theresidue is recrystallized from methanol/ethyl acetate/hexane and 2.1 gof(3S,4R)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-{(2S-2-N-benzyloxycarbonylamino-2-diphenylmethoxycarbonyl-ethyl)-phenoxy}-azetidin-2-one is obtained as white solid.

910 mg of(3S,4R)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-{(2S-2-N-benzyloxycarbonylamino-2-diphenylmethoxycarbonyl-ethyl)-phenoxy}-azetidin-2-oneis hydrogenated with 2 g of 10% palladium on activated carbon in amixture of ethyl acetate (50 ml), THF (50 ml) and ethanol (20 ml) at 50psi hydrogen pressure at room temperature for 4 hours. The solid isfiltered and washed with ethyl acetate (3×20 ml). The solid is extractedwith a mixture of water/acetonitrile (4:6) (2×50 ml). After removal ofsolvent, the resulting solid is washed with acetonitril and 265 mg ofthe title compound is obtained as white solid.

Yield: 45%, m.p.: 161-162° C., FAB-MS: 551 (MH⁺), calcd for C₃₀ H₃₈ N₄O₆ 550

¹ H NMR (DMSO-d₆), δ (ppm): 0.7-1.8 (13H, m), 2.35-2.50 (2H, m), 2.7-2.9(2H, m), 3.2-3.4 (2H, m), 4.35 (1H, m), 5.27 (1H, dd, J=8, 3 Hz), 5.65(1H, d, J=3 Hz), 6.82 (2H, m), 7.05-7.30 (7H, m), 7.94 (1H, d, J=8 Hz),8.64 (1H, d, J=8 Hz), 9.28 (1H, s).

IR (KBr, cm⁻¹): 3400, 3290, 2925, 1771, 1643, 1555, 1506, 1396, 1230.

EXAMPLE 5(3S,4SR)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-phenylthio-azetidin-2-one(5)

To a solution of thiophenol (149 mg, 1.36 mmol) in THF (5 ml), water (5ml) and 1 N NaOH (1.2 ml),(3S,4S)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one(387 mg, 0.9 mmol) in acetone (10 ml) and THF (5 ml) is added at 5° C.The mixture is stirred at 5° C. for 1 hour and then at room temperaturefor 1 hour. After removal of solvent, the residue is dissolved in ethylacetate, washed with water, brine and dried over sodium sulphate. Afterremoval of solvent, the residue is purified by recrystallization fromTHF-ethyl acetate-hexane and 164 mg of title compound is obtained.

Yield: 38%, m.p.: 198-200° C., FAB-MS: 480 (MH⁺), calcd for C₂₇ H₃₃ N₃O₃ S 479

¹ H NMR (DMSO-d₆), δ (ppm): 0.7-17 (13H, m), 2.45 (2H, m), 2.80 (2H, m),4.34 (0.85H, m), 4.45 (0.15H, m), 4.54 (0.85H, dd, J=8.5, 2.0 Hz), 4.92(0.85H, d, J=2.0 Hz), 5.25-5.35 (0.3H, m), 7.10-7.50 (10H, m), 7.98(0.15H, d, J=8.1 Hz), 8.05 (0.85H, d, J=8.1 Hz), 8.71 (0.85H, d, J=8.6Hz), 8.83 (0.15H, d, J=8.6 Hz), 9.00 (1H, s).

IR (KBr, cm⁻¹): 3270, 2905, 1763, 1735, 1634, 1523, 1436, 1367, 1222.

EXAMPLE 6(3S,4SR)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-phenylsulfonyl-azetidin-2-one(6)

A mixture of(3S,4SR)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-phenylthio-azetidin-2-one(100 mg, 0.208 mmol) obtained in example 5, and KMnO₄ (50 mg, 0.32 mmol)in acetic acid (10 ml) and H₂ O (2 ml) is stirred at 5° C. for 1 hourand then room temperature for 1 hour. One drop of H₂ O₂ (30% aq) isadded. The reaction mixture is partitioned between ethyl acetate andwater, the organic layer is washed with water, saturated NaHCO₃, water,brine and dried over Na₂ SO₄. After removal of the solvent, solid iswashed with ether and 78 mg of the title compound is obtained. Yield:73%, m.p.: 170° C. (dec.), FAB-MS: 512 (MH⁺), calcd for C₂₇ H₃₃ N₃ O₅ S511

¹ H NMR (DMSO-d₆), δ (ppm): 0.6-1.7 (13H, m), 2.45 (2H, m), 2.80 (2H,m), 4.30 (0.85H, m), 4.50 (0.15H, m), 4.87 (0.85H, dd, J=8.2 & 2.1 Hz),4.95 (0.85H, d, J=2.1 Hz), 5.20 (0.15H, d, J=4.6 Hz), 5.51 (0.15H, m),7.22 (5H, m), 7.60-8.00 (5H, m), 8.05 (1H, d, J=8.3 Hz), 8.48 (0.15H, d,J=8.4 Hz), 8.71 (0.85H, d, J=8.4 Hz), 9.31 (0.85H, s), 9.40 (0.15H, s).

IR (KBr, cm⁻¹): 3280, 2905, 1779, 1640, 1517, 1440, 1301.

EXAMPLE 7(3S,4S)-3-{2S-2-(benzylaminocarbonyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one(7)

(3S,4S)-3-{2S-2-(benzyloxycarbonyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one(from example 1) (216 mg, 0.5 mmol) is hydrogenated with 400 mg of 10%palladium on activated carbon in ethyl acetate (15 ml) and THF (7 ml) at50 psi hydrogen pressure at room temperature for 3 hours. After removalof catalyst by filtration, desubstituted(3S,4S)-3-(2S-2-amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-onein ethyl acetate/THF is cooled to -15° C. and then benzyl isocyanate(106 mg, 0.8 mmol) is added. The reaction mixture is stirred at -10 to0° C. for 1 hour and room temperature for 1 hour. After removal ofsolvent, the residue is dissolved in ethyl acetate, washed with coldsaturated NaHCO₃ solution, water, brine and dried over sodium sulphate.After removal of solvent, the residue is purified by silica gel columnchromatography using hexane-ethyl acetate (1:2) as eluent and the titlecompound is obtained.

Yield: 74%, m.p.: 192-194° C., FAB-MS: 431 (MH⁺), calcd for C₂₂ H₃₀ N₄O₅ 430

¹ H NMR (DMSO-₆), δ (ppm): 0.7-1.8 (13H, m), 2.07 (3H, s), 4.15-4.30(3H, m), 4.64 (1H, d, J=8.5 Hz), 5.74 (1H, s), 6.15 (1H, d, J=8.6 Hz),6.46 (1H, m), 4.20-4.35 (5H, m), 8.71 (1H, d, J=8.5 Hz), 9.16 (1H, s).

IR (KBr, cm⁻¹): 3325, 2905, 1789, 1732, 1653, 1628, 1554, 1526, 1440,1357, 1223.

EXAMPLE 8(3S,4S)-3-{2S-2-(phenylethenylsulfonyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one(8)

(3S,4S)-3-{2S-2-(benzyloxycarbonyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one(from example 1) (216 mg, 0.5 mmol) is hydrogenated with 400 mg of 10%palladium on activated carbon in ethyl acetate (15 ml) and THF (7 ml) at50 psi hydrogen pressure at room temperature for 3 hours. After removalof catalyst by filtration, desubstituted(3S,4S)-3-(2S-2-amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-onein ethyl acetate/THF is cooled to -15° C. and then triethylamine (50 mg,0.5 mmol) and benzyl isocyanate (106 mg, 0.8 mmol) is added. Thereaction mixture is stirred at -10 to 0° C. for 1 hour and at 5° C.overnight. After removal of solvent, the residue is dissolved in ethylacetate, washed with cold saturated NaHCO₃ solution, water, brine anddried over sodium sulphate. After removal of solvent, the residue ispurified by silica gel column chromatography using hexane-ethyl acetate(1:1) as eluent and the title compound is obtained.

Yield: 35%, m.p.: 77° C. (dec.), FAB-MS: 464 (MH⁺), calcd for C₂₂ H₂₉ N₃O₆ S 463

¹ H NMR (DMSO-d₆), δ (ppm): 0.7-1.8 (13H, m), 2.02 (3H, s), 3.70-3.85(1H, m), 4.61 (1H, d, J=7.6 Hz), 5.54 (1H, s), 6.99 (1H, d, J=15.5 Hz),7.32 (1H, d, J=15.5 Hz), 7.40-7.50 (3H, m), 7.60-7.70 (2H, m), 7.82 (1H,d, J=7.6 Hz), 8.80 (1H, d, J=8.0 Hz), 9.18 (1H, s).

IR (KBr, cm⁻¹): 3295, 2905, 1778, 1744, 1659, 1521, 1441, 1317, 1222.

EXAMPLE 9(3S,4S)-3-(2S-2-benzyloxycarbonylamino-2-cyclohexylmethyl-acetamido)-4-(3-methyl-phenoxy)-azetidin-2-one(9A) and (3S,4R)-3-(2S-2-benzyloxycarbonylamino-2-cyclohexylmethyl-acetamido)-4-(3-methyl-phenoxy)-azetidin-2-one(9B)

To a solution of 3-methyl-phenol (81 mg, 0.75 mmole) in acetone (2 ml)and 1N NaOH (0.6 ml, 0.6 mmole),(3S,4S)-3-(2S-2-benzyloxycarbonylamino-2-cyclohexylmethyl-acetamido)-4-acetoxy-azetidin-2-one(216 mg, 0.5 mmole) in THF (4 ml) and H₂ O (1 ml) is added at 0° C. Themixture is stirred at 0° C. for 1 hour and then at room temperature for30 min. After removal of solvent, the residue is dissolved in ethylacetate, washed with water, brine and dried over sodium sulfate. Afterremoval of solvent, the residue is purified by silica gel columnchromatography using hexane-ethyl acetate as eluent. 110 mg of(3S4S)-3-(2S-2-benzyloxycarbonylamino-2-cyclohexyl-methyl-acetamido)-4-(3-methyl-phenoxy)-azetidin-2-one(9A) and 40 mg of(3S,4R)-3-(2S-2-benzyloxycarbonylamino-2-cyclohexylmethyl-acetamido)-4-(3-methyl-phenoxy)-azetidin-2-one(9B) are obtained.

For (9A)

Yield: 46%

m.p.: 184-185.5° C.

¹ H-NMR (DMSO-d₆), δ (ppm): 0.7-1.8 (13H, m), 2.26 (3H,s), 4.0-4.2 (1H,m), 4.64 (1H, d, J=8.5 Hz), 5.05 (2H, m), 5.50 (1H,s), 6.6-6.7 (2H, m),6.83 (1H,d, J=7.3 Hz), 7.1-7.4 (6H,m), 7.52 (1H,d, J=8 Hz), 8.82 (1H,d,J=8.5 Hz), 9.28 (1H, s).

For (9B)

Yield: 17%

m.p.: 178-179° C.

¹ H-NMR (DMSO-d₆) δ (ppm): 0.7-1.8 (13H, m), 2.24 (3H,s), 4.0-4.2 (1H,m), 5.01 (2H, m), 5.33 (1H,m), 5.68 (1H,d, J=3.7 Hz), 6.6-6.85 (3H,m),7.1-7.4 (7H, m), 8.61 (1H,d, J=9.2 Hz), 9.23 (1H, s).

EXAMPLE 10 (3S,4S)-3-{2S-2-3-(pyridin-4-yl)propenoyl!amino-2-cyclohexylmethyl-acetamido}-4-phenoxy-azetidin-2-one(10)

The title compound was synthesized by the reaction of succinimidyl3-(pyridin-4-yl) propanoic acid with(3S,4S)-3-(2S-2-amino-2-cyclohexylmethyl-acetamido)-4-phenoxy-azetidin-2-onein DMF.

Yield: 43%

m.p.: 145-147° C.

¹ H-NMR (DMSO-d₆), δ (ppm): 0.7-1.8 (13H, m), 4.35-3.50 (1H, m), 4.66(1H, d, J=8.3 Hz), 5.55 (1H,s), 6.86-7.55 (9H, m), 8.54 (1H,d, J=8.0Hz), 8.60 and 8.65 (2H,2s), 8.93 (1H,d, J=8.4 Hz), 9.31 (1H, s).

EXAMPLE 11 (3S,4S)-3-{2S-2-3-(pyridin-3-yl)propenoyl!amino-2-cyclohexylmethyl-acetamido}-4-phenoxy-azetidin-2-one(11)

The title compound was synthesized by the reaction of succinimidyl3-(pyridin-3-yl) propenoic acid with(3S,4S)-3-(2S-2-amino-2-cyclohexylmethyl-acetamido)-4-phenoxy-azetidin-2-onein DMF.

Yield: 47%

m.p.: 148-150° C.

¹ H-NMR (DMSO-d₆), δ (ppm): 0.3-1.8(13H,m), 4.42-4.54 (1H, m), 4.67 (1H,d, J=8.0 Hz), 5.55 (1H,s), 6.82-7.56 (8H, m), 7.99 (1H,d, J=7.9 Hz),8.45 (1H,d, J=8.0 Hz), 8.56 (1H,d, J=4.7 Hz), 8.77 (1H,s), 8.92 (1H, d,J=8.5 Hz), 9.31 (1H, s).

Testing of inhibitors for inhibition of Cathepsin B and L

TEST EXAMPLE 1

In vitro assay procedure for cathepsin B

The compounds of formula I are tested for inhibition of cathepsin Busing the known method (A. J. Barret et al., Biochem. J. 1982, 201,189-198). To 170 μl of an enzyme-buffer mixture (enzyme: recombinant ratcathepsin B, diluted to give approximate 10 Fluorescence units/min,buffer: 56 mM sodium acetate, 1.124 mM EDTA, 10 mM DTT, pH 5.1) 10 μL ofinhibitor (dissolved in DMSO) is added. After 10 min of incubation atroom temperature, a 20 μl of 5 mM substrate (N-CBZ-Phe-Arg-AMC,dissolved in DMSO) is added to initiate reaction. Reading is followed upfor 10 min on a Fluoroskan fluorescence reader (excitation at 380 nm,emission at 460 nm).

A plot of percentage of inhibition vs inhibitor concentration isobtained, and IC₅₀ is determined using a linear regression calculation(concentration of inhibitor which will give 50% inhibition).

TEST EXAMPLE 2

In vitro assay procedure for cathepsin L

To 170 μl of enzyme-buffer mixture (enzyme: recombinant rat cathepsin L,diluted to give approximate 15 Fluorescence units/min, buffer: 58.8 mMsodium citrate, 1.18 mM EDTA, 235 mM sodium chloride, 5 mM DTT, pH 5.0)10 μL of inhibitor (dissolved in DMSO) is added. After 10 min ofincubation at room temperature, 20 μl of 1 mM substrate(N-CBZ-Phe-Arg-AMC, dissolved in DMSO) is added to initiate reaction.Reading is followed up for 10 min on a Fluoroskan fluorescence reader(excitation at 380 nm emission at 460 nm).

A plot of percentage of inhibition vs inhibitor concentration isobtained, and IC₅₀ is determined using a linear regression calculation(concentration of inhibitor which will give 50% inhibition).

                  TABLE 1    ______________________________________    In vitro inhibitory activity of monobactam compounds on cysteine    proteases.                IC.sub.50 (μM)    Example No.   Cathepsin B                            Cathepsin L    ______________________________________     1            8.71      0.78     2            11.6      2.32     3            34        1.82     4            9.2       1.8     5            10.4      0.016     6            29        0.078     7            11.6      2.30     8            11        2.16     9A           6.9       0.083     9B           0.25      0.003    10            1         0.4    11            2.2       0.43    ______________________________________

TEST EXAMPLE 3

In vitro stability test in rat plasma

The testing compound is added to Rat plasma/phosphate buffer (pH=7.4) at37° C. (the final concentration is 200 μg/ml) and the resulting solutionkept at 37° C. Samples are taken at 0, 0.5, 1, 2, 4, and 6 hours. 500 μlof sample is taken in duplicate for each time. To the 500 μl of thesample, 500 μl of ice-cold acetonitrile is added to precipitate theprotein, and the product is then vortexed for 30 seconds and centrifugedat 5000 rpm for 10 mins. The supernatant is removed and to it is added2.0 ml of methylene chloride. The mixture is vortexed for 30 seconds andthen centrifuged at 5000 rpm for 10 mins. The upper layer is directlyinjected onto the HPLC for analysis. The results are shown in theFIGURE.

TEST EXAMPLE 4

In vivo inhibition test for cathepsin B and L

The in vivo inhibition of cathepsin B and L are tested according to theknown method (T. Towatari et al, FEBS, 1991, 280, 311-315). Inhibitor isinjected intraperitoneally into rodents as a solution in salinecontaining DMSO or DMSO:PEG400 (1:1) at the doses indicated in Table 2.The rodents are killed after 6 hours, and the liver is perfused withice-cold saline, and chilled on ice. Sample of 4 g of liver arehomogenized in 7 volumes of 0.25 M sucrose. The homogenate iscentrifuged at 800 g for 15 min. and the supernatant is centrifuged at12,000 g for 30 min. The precipitate (crude mitochondrial-lysosomalfraction; ML fraction) is suspended in 2 ml of 0.05 M acetate buffer, pH5.0, and then freeze-thawed for measurements of cathepsin B and L.

                  TABLE 2    ______________________________________    In vivo inhibition of the inhibitors for cathepsin B and L.                Dosage  Inhibition.sup.a    Compd.    Species (mg/kg)   Cathepsin B                                        Cathepsin L    ______________________________________    Ref. Compd..sup.b              rat     30        37%     30%              rat     70        56%     55%    Compd. 3  mouse   50        65%     55%    ______________________________________     .sup.a Values are means for 3 animals.     .sup.b Reference compound is (3S,     4S)3-{N(3-phenyl-propionoyl)-L-phenylalanyl}amino4-(4-(2S-2-amino-2-carbo    y ethyl)phenoxyazetidin-2-one.

Although the compounds, the methods of treatment and the methods ofmaking the compounds in accordance with the present invention have beendescribed in connection with preferred embodiments, it will beappreciated by those skilled in the art that modifications notspecifically described may be made without departing from the spirit andscope of the invention defined in the following claims.

We claim:
 1. A 4-substituted-3-(2-amino-2-cycloalkyl methyl)-acetamidoazetidin-2-one compound of formula I, or a pharmaceutically acceptablesalt thereof: ##STR10## wherein n is 1, 2 or 3;R₁ ishydrogen; or --SO₃ ⁻M⁺ wherein M is a hydrogen atom, a metal ion which is selected fromsodium, potassium, magnesium, and calcium, or N⁺ (R₄)₄ wherein R₄ is aC₁ -C₆ alkyl group; R₂ is(a) a group --OCOR₅ wherein R₅ is(i) a C₁ -C₆alkyl group, (ii) a C₂ -C₆ alkenyl group, (iii) a C₂ -C₆ alkynyl group,(iv) a C₃ -C₆ cycloalkyl group, (v) a phenyl group, (vi) a naphthylgroup, or (vii) a monocyclic or bicyclic heterocyclic group, which group(i), (ii), (iii), (iv), (v), (vi), or (vii) is unsubstituted orsubstituted by 1, 2 or 3 substituents independently selectedfromhydroxy, halogen, carboxy, C₁ -C₄ alkyl (which is unsubstituted orsubstituted at least once with carboxy and/or amino), C₁ -C₂ alkoxy,amino, cyano, and phenyl and monocyclic or bicyclic heterocyclic groups,which phenyl and heterocyclic groups are unsubstituted or substituted by1 or 2 substituents independently selected from hydroxy, halogen,carboxy, C₁ -C₄ alkyl, C₁ -C₂ alkoxy, amino, and cyano; or (b) a group--XR₅ wherein X is selected from the group consisting of O, S, SO, andSO₂, and R₅ is as defined above; R₃ is hydrogen, --COOR₅, --COR₅, --SO₂R₅, or --COR₁₄ wherein R₅ is as defined above and R₁₄ is amino groupwhich is unsubstituted or substituted at least once with C₁ -C₆ alkylgroup which is unsubstituted or substituted at least once with 1 or 2substitutents selected from hydroxy, halogen, cyano, amino, heterocycle,and phenyl (wherein the heterocycle or phenyl is unsubstituted orsubstituted at least once by 1 or 2 substituents selected from halogen,hydroxy, cyano, carboxy and amino),wherein the heterocyclic group asdefined in R₅ or R₁₄ are C₂ -C₉ monocyclic or bicyclic heterocyclicgroup which have 1-3 heteroatoms selected from nitrogen, sulfur andoxygen.
 2. A 4-substituted-3-(2-amino-2-cycloalkyl methyl)-acetamidoazetidin-2-one compound of formula I, or a pharmaceutically acceptablesalt thereof: ##STR11## wherein n is 1, 2 or 3R₁ ishydrogen; --SO₃ ⁻ M⁺wherein M is a hydrogen atom, a metal ion which is selected from sodium,potassium, magnesium, or calcium, or N⁺ (R₄)₄ wherein R₄ is a C₁ -C₆alkyl group; R₂ is--OCOR₅ wherein R₅ is(i) a C₁ -C₆ alkyl group which isunsubstituted or substituted at least once by 1 or 2 substitutentsselected from hydroxy, halogen, and amino; or (ii) a phenyl group whichis unsubstituted or substituted at least once by 1-3 substituentsselected from hydroxy, halogen, C₁ -C₄ alkyl group, C₁ -C₂ alkoxy group,and cyano; --XR₆ wherein X is O, S, SO, or SO₂ ; R₆ is(i) a C₁ -C₆ alkylgroup which is unsubstituted or substituted at least once by 1 or 2substitutents selected from hydroxy, halogen, amino or phenyl; or (ii) aphenyl group which is unsubstituted or substituted at least once by 1-3substituents selected from hydroxy, halogen, carboxy, and C₁ -C₄ alkylgroup which is unsubstituted or substituted at least once with carboxy,amino or both, C₁ -C₂ alkoxy group, cyano or heterocycle group; R₃ishydrogen; --COOR₇ wherein R₇ is a C₁ -C₆ alkyl group which isunsubstituted or substituted at least once with phenyl and/orheterocycle group; --COR₈ wherein R₈ is(i) a C₁ -C₆ alkyl group which isunsubstituted or substituted at least once by 1 or 2 substitutentsselected from hydroxy, halogen, cyano, amino, heterocycle, and phenyl,wherein said heterocycle or phenyl is unsubstituted or substituted atleast once by 1 or 2 substituents selected from halogen, hydroxy, cyano,carboxy and amino; or (ii) an amino group which is unsubstituted orsubstituted at least once with C₁ -C₆ alkyl group which is unsubstitutedor substituted at least once by 1 or 2 substitutents selected fromhydroxy, halogen, cyano, amino, heterocycle and phenyl, wherein saidheterocycle or phenyl is unsubstituted or substituted at least once by 1or 2 substituents selected from halogen, hydroxy, cyano, carboxy andamino; or --SO₂ R₉ wherein R₉ is(i) a C₁ -C₆ alkyl group which isunsubstituted or substituted at least once with heterocycle and/orphenyl; or (ii) a C₂ -C₄ alkenyl group which is unsubstituted orsubstituted at least once with heterocycle and/or phenyl,whereinheterocyclic group as defined in R₆, R₇, R₈ or R₉ are C₂ -C₉ mono orbicyclic heterocyclic group which have 1-3 heteroatoms selected fromnitrogen, sulfur and oxygen.
 3. A compound or salt as recited in claim2, wherein halogen atoms as substitutents in R₅, R₆, or R₉ are fluorine,chlorine or bromine.
 4. A method of regulating a cysteine proteaseselected from the group consisting of Cathepsin B and Cathepsin L in apatient in need of arthritis treatment, comprising administering to saidpatient a compound or salt as recited in claim 1 in an amount which iseffective for treating arthritis.
 5. A compound selected from the groupconsistingof:(3S,4S)-3-(2S-2-benzyloxycarbonylamino-2-cyclohexylmethyl-acetamido)-4-acetoxy-azetidin-2-one;(3S,4S)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one;(3S,4S)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-{4-(2S-2-amino-2-carboxy-ethyl)-phenoxy}-azetidin-2-one;(3S,4R)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-{4-(2S-2-amino-2-carboxy-ethyl)-phenoxy}-azetidin-2-one;(3S,4SR)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-phenylthio-azetidin-2-one;(3S,4SR)-3-{2S-2-(3-phenylpropionoyl)amino-2-cyclohexylmethyl-acetamido}-4-phenylsulfonyl-azetidin-2-one;(3S,4S)-3-{2S-2-(benzylaminocarbonyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one;(3S,4S)-3-{2S-2-(phenylethenylsulfonyl)amino-2-cyclohexylmethyl-acetamido}-4-acetoxy-azetidin-2-one;(3S,4S)-3-(2S-2-benzyloxycarbonylamino-2-cyclohexylmethyl-acetamido)-4-(3-methyl-phenoxy)-azetidin-2-one;(3S,4R)-3-(2S-2-benzyloxycarbonylamino-2-cyclohexylmethyl-acetamido)-4-(3-methyl-phenoxy)-azetidin-2-one;(3S,4S)-3-{2S-2-3-(pyridin-4-yl)propenoyl!amino-2-cyclohexylmethyl-acetamido}-4-phenoxy-azetidin-2-one;and (3S,4S)-3-{2S-2-3-(pyridin-3-yl)propenoyl!amino-2-cyclohexylmethyl-acetamido}-4-phenoxy-azetidin-2-one.6. A compound or salt as recited in claim 2, wherein C₁ -C₄ alkyl groupas substituents in R₅, R₆, or R₉ are branched or straight alkyl chainselected from methyl, ethyl, propyl, 2-methyl propyl, butyl and1,1-dimethyl ethyl.
 7. A compound or salt as recited in claim 2, whereinsaid compound is the L isomer of the asymmetric carbon having thecycloalkyl methyl and amino substituents thereon.
 8. A compound or saltas recited in claim 2, wherein said compound is selected from the 3S, 4Sand 3S, 4R diastereoisomers and the 3S, 4SR racemate of the C3 and C4asymmetric carbon of the azetidinone nucleus.
 9. A pharmaceuticalcomposition comprising a compound or salt as recited in claim 1 and apharmaceutically acceptable carrier.
 10. A salt as recited in claim 2,wherein said salt comprises a component selected from sodium, potassium,magnesium, calcium, hydrogen chloride, tartaric acid, succinic acid,fumaric acid and p-toluenesulfonic acid.
 11. A compound or salt asrecited in claim 8, wherein said compound is selected from the isomersin which C3 and C4 are cis to each other.
 12. A compound or salt asrecited in claim 11, wherein C₁ -C₆ alkyl group as substituents in R₄,R₅, R₆, R₈, or R₉ are selected from methyl, ethyl, propyl,1-methylethyl, butyl, 1-methylpropyl, 2-methylprop-1-yl,2-methylprop-2-yl, pentyl, 3-methylbutyl, and hexyl.
 13. A compound orsalt as recited in claim 4, wherein C₂ -C₄ alkenyl group as defined inR₉ are selected from ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, and3-butenyl.
 14. A compound or salt as recited in claim 5, whereinheterocyclic group as defined in R₆ R₇, R₈ or R₉ are selected fromthiophene, pyridine, 1,2,3-triazole, 1,2,4-triazole, quinoline,benzofuran, benzothiophene, morpholine, thiomorpholine, piperizine, andpiperidine.